Experiments have been designed to elucidate the exact nature of membrane lipid asymmetry in animal cell systems with emphasis on the respective roles of phospholipid side chains, polar head groups, and cholesterol. To elicit the kinds of information needed to establish the physical properties of each monolayer, membrane-enveloped viruses and phagocytized latex beads (phagosomes) are being used as sources of right side out and inside out membranes for ESR spin labeling studies. Preliminary evidence with a new amphiteric spin label whose polar group restricts penetration to the outer half of the membrane bilayer shows that the exposed monolayers of right side out and inside out membrane preparations have different physical states. When disrupted or unsealed membrane preparations are examined, the characteristic temperatures defining the physical states of both monolayers are revealed. Future projects will examine the basis and the role of vertical physical asymmetry in biological membranes.